Not the mature 56 kDa lipoprotein lipase protein but a 37 kDa protein co-purifying with the lipase mediates the binding of low density lipoproteins to J774 macrophages.
Lipoprotein lipase (LPL) purified from bovine milk showed variable abilities to stimulate the binding of low density lipoprotein (LDL) to J774 macrophages. The presence of a 37 kDa protein in the LPL sample seemed to be of importance for its stimulatory capacity. In order to investigate this, we isolated LPL from bovine milk via heparin Sepharose chromatography using a continuous salt gradient. Fractions containing the 37 kDa protein (as shown by SDS/PAGE under reducing conditions) eluted first from the column, followed by the 56 kDa LPL protein. The LPL enzymatic activity co-eluted with the 56 kDa protein, whereas the amount of 37 kDa protein fully paralleled the stimulatory effect on the binding of LDL to J774 cells. Samples not containing the 37 kDa protein were far less effective in stimulating the binding. Western blotting using a monoclonal antibody 5D2 against amino acids 396-405 in the carboxy-terminal domain of LPL, showed that the 37 kDa protein may be the C-terminal domain of LPL, presumably generated by proteolytic degradation of the mature LPL protein by milk proteases during its isolation. Furthermore, the functional mass of LPL for stimulation of the binding of LDL, as determined by radiation inactivation, was shown to be 30.9+/-1.8 kDa. We therefore suggest that cleavage of LPL at protease-sensitive sites causes a conformational change, generating an LPL protein which is more effective in mediating the binding and uptake of lipoproteins by cells.
Hendriks
W L
WL
TNO Prevention and Health, Gaubius Laboratory, Zernikedreef 9, 2333 CK Leiden, P.O. Box 2215, 2301 CE Leiden, The Netherlands.
Van Vark
L C
LC
Schoonderwoerd
K
K
Jansen
H
H
Havekes
L M
LM
D000818:Animals; D015153:Blotting, Western; D002417:Cattle; D002478:Cells, Cultured; D002850:Chromatography, Gel; D006801:Humans; D008071:Lipoprotein Lipase / Q000378:metabolism*; D008077:Lipoproteins, LDL / Q000378:metabolism*; D008264:Macrophages / Q000378:metabolism*; D051379:Mice; D008892:Milk / Q000201:enzymology; D008970:Molecular Weight
D008077:Lipoproteins, LDL; D008071:Lipoprotein Lipase